A transforming growth factor-beta antagonist unmasks the neuroprotective role of this endogenous cytokine in excitotoxic and ischemic brain injury.

Various studies describe increased concentrations of transforming growth factor-beta (TGF-beta) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-beta after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-beta produced after an episode of cerebral ischemia by injecting a soluble TGF-beta type II receptor fused with the Fc region of a human immunoglobulin (TbetaRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-beta. Then, the authors tested its ability to reverse the effect of TGF-beta1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 microg/mL of TbetaRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-beta1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-beta1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-beta1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 microg of TbetaRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-beta1 mRNA. Furthermore, when added to the excitotoxin, TbetaRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TbetaRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-beta1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 +/- 9.5 versus 152.8 +/- 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-beta in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-beta1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.

Patterns of messenger RNA expression for alpha1-adrenoceptor subtypes in human corpus cavernosum.

PURPOSE: The present study evaluated the expression of alpha1-adrenoceptor subtypes in human corpus cavernosum. MATERIALS AND METHODS: The mRNA encoding alpha1a, alpha1b and alpha1d subtypes were assessed by RNA-directed complementary cDNA synthesis followed by Taq DNA amplification. The level of alpha1 mRNA was calculated in arbitrary optical density units and normalized with respect to the length of the respective cDNA fragments. RESULTS: We found that alpha1a, alpha1b and alpha1d adrenoceptor subtypes are expressed in human corpus cavernosum, with a predominant expression of the alpha1a subtype. CONCLUSION: These results suggest that alpha1a-adrenoceptor subtype is important and that understanding the biochemical and functional characteristics of this subtype may lead to the development of specific antagonists in the treatment of impotence.

Antiproliferative effect of Pygeum africanum extract on rat prostatic fibroblasts.

The effect of a Pygeum africanum extract (Tadenan) (Pa), used in the treatment of micturition disorders associated with BPH, has been examined on the proliferation of rat prostatic stromal cells stimulated by different growth factors. EGF, bFGF, and IGF-I but not KGF are mitogenic for prostatic fibroblasts in culture. Pygeum africanum inhibits both basal and stimulated growth with IC50 values of 4.5, 7.7 and 12.6 micrograms./ml. for EGF, IGF-I and bFGF, respectively, compared to 14.4 micrograms./ml. for untreated cells, the inhibition being stronger towards EGF. Pygeum africanum inhibited the proliferation induced by TPA or PDBu in a concentration-dependent manner with IC50 values of 12.4 and 8.1 micrograms./ml. respectively. The antiproliferative effects of Pa were not ascribed to cytotoxicity. These results show that Pygeum africanum is a potent inhibitor of rat prostatic fibroblast proliferation in response to direct activators of protein kinase C, the defined growth factors bFGF, EGF and IGF-I, and the complex mixture of mitogens in serum depending on the concentration used. PKC activation appears to be an important growth factor-mediated signal transduction for this agent. These data suggest that therapeutic effect of Pygeum africanum may be due at least in part to the inhibition of growth factors responsible for the prostatic overgrowth in man.

Effects of transection of the spinal cord in the rat: cystometric and autoradiographic studies.

Cystometric studies and autoradiographic experiments were performed in the rat to determine the urodynamic parameters and the cholinergic muscarinic binding properties of detrusor in control animals and those with complete spinal cord transection. After an unexplored spinal shock phase, the reactivity of the bladder was studied at 2, 5 and 9 weeks following transection. The chief modifications caused by spinal cord injury on the cystomanometric parameters were an increase of the maximal amplitude of contraction and a decrease of the pressure threshold. This correlated well with the results obtained in the autoradiographic studies, in which the density of muscarinic receptors in transected rats increased by 80% and 60% in the vesical sections after 2 and 5 weeks. The density of these muscarinic receptors sites returned to control levels 9 weeks after section.

Non-adrenergic binding sites for the "alpha 2-antagonist" [3H]idazoxan in the rabbit urethral smooth muscle. Pharmacological and biochemical characterization.

In the present study, pharmacological and biochemical binding characteristics of [3H]idazoxan, an originally thought alpha 2-adrenoceptor antagonist, have been determined in smooth muscle of rabbit urethra. It is shown that [3H]idazoxan labels with high affinity non-adrenergic binding sites. Specific binding of [3H]idazoxan is inhibited by compounds possessing an imidazoline or a guanidinium moiety whereas phenylethanolamines and classical alpha 2-antagonists are ineffective competitors which suggests an imidazoline-preferring binding site. However, imidazolidines such as clonidine and paminoclonidine are poorly effective, which differs considerably from pharmacological characteristics of imidazoline binding sites previously reported in the central nervous system. In addition, it is shown that K+ and Mn2+ inhibit [3H]idazoxan binding in a competitive and non-competitive manner, respectively. Other cations such as Na+, Li+ and Mg2+ have no significant effect. It is shown that K+ accelerates the dissociation of [3H]idazoxan binding while Mn2+ does not produce any modification. These results suggest that K+ may bind to an allosteric site, while Mn2+ may bind with a membrane component susceptible to alter [3H]idazoxan binding sites.

Non-adrenergic binding sites for the "alpha 2-antagonist" [3H] idazoxan in rabbit urethral smooth muscle.

In the present study, we have provided evidence that [3H] rauwolscine and [3H] idazoxan bind to different sites in rabbit urethra. The [3H] idazoxan capacity and affinity was 215 +/- 14 fmol/mg protein and 1.59 +/- 0.16 nM while [3H] rauwolscine binding parameters were 45.9 +/- 3.4 fmol/mg protein and 2.39 +/- 0.27 nM. [3H] idazoxan specific binding was inhibited only by compounds possessing an imidazoli(di)ne or a guanidinium moiety, while [3H] rauwolscine specific binding was inhibited by phenylethanolamines and classical alpha 2-antagonists. [3H] idazoxan was inhibited by KCl in a competitive and by MnCl2 in a non-competitive way, while other cations such as Na+, Li+ and Mg2+ did not inhibit [3H] idazoxan binding. Moreover, we investigated the regional distribution of [3H] idazoxan and [3H] rauwolscine along the rabbit urethra using quantitative autoradiography. Analysis of the films revealed a different distribution of these two binding sites on the urethral sections.

Amiloride interacts with [3H]idazoxan and [3H]rauwolscine binding sites in rabbit urethra.

Amiloride and its analogues (N-ethylisopropylamiloride and banzamil) interact more potently with [3H]idazoxan binding sites (nM range) than with [3H]rauwolscine binding sites (microM range) in the rabbit urethra. The binding of both radioligands was competitivelly inhibited by amiloride, with increased KD values and no change in their binding capacity. Interestingly, amiloride was a potent inhibitor at [3H]idazoxan binding sites in rabbit urethral smooth muscle at concentrations far below those required to inhibit the Na+/H+ exchanger or electrogenic pump.

Evidence for non-adrenergic binding sites for [3H]idazoxan in the smooth muscle of rabbit urethra.

The binding characteristics of [3H]idazoxan and [3H]rauwolscine, two potent alpha 2-adrenoceptor antagonists, were compared in the rabbit urethral smooth muscle. The maximal binding capacity was 6 times higher for [3H]idazoxan than for [3H]rauwolscine in male rabbits. No difference was observed for these radioligands in female rabbits. There were marked differences in the ability of alpha 2-adrenergic compounds to inhibit [3H]idazoxan and [3H]rauwolscine binding. These results were consistent with the existence of non-alpha 2-adrenoceptor sites for [3H]idazoxan in the rabbit urethral smooth muscle.

Alpha 1- and alpha 2-adrenoceptors in the smooth muscle of male and female rabbit urethra.

[3H]Prazosin and [3H]rauwolscine, specific alpha 1- and alpha 2-antagonists respectively, were used to label alpha-adrenoceptors in membranes from male and female rabbit urethra. In the male rabbit, [3H]prazosin bound with high affinity (Kd = 0.56 nM) to a single population of sites with a capacity of 73 fmol/mg protein. [3H]Rauwolscine bound with a lower affinity (Kd = 2.24 nM) to another single class of sites with a capacity of 41 fmol/mg protein. The order of potencies of various adrenergic compounds in inhibiting radioligand binding suggested that [3H]prazosin and [3H]rauwolscine interacted in the urethra with sites having the characteristics of alpha 1- and alpha 2-adrenoceptors, respectively. In addition, studies on the female rabbit urethra showed that alpha 2-adrenoceptor density and affinity were respectively 6 times higher and 2 times lower than in the male. No significant sex difference was observed for urethral alpha 1-adrenoceptors.

Alteration of membrane permeability in Bacillus subtilis by clofoctol.

When Bacillus subtilis was treated with a bacteriostatic concentration of clofoctol [2-(2,4-dichlorobenzyl)-4-(tetramethyl-1,1,3,3-butyl)phenol], UV-absorbing material was released. Electron micrographs showed evidence of physical alteration of the bacterial envelope. The uptake of [14C]glutamate was strongly inhibited by clofoctol, and preloaded glutamate was found to leak from the bacteria. Clofoctol caused an immediate and dramatic decrease in the amount of intracellular ATP. This was neither the consequence of the stimulation of an ATPase, nor of the inhibition of bacterial respiration. Both the proton gradient and the potential gradient across the cytoplasmic membrane collapsed and this inhibition of energy metabolism was sufficient to account for the inhibition of growth by clofoctol. At the same bacteriostatic concentration complete permeabilization of the bacteria occurred.

Action of clofoctol on bacterial cell wall synthesis.

Clofoctol inhibits in vivo cell wall synthesis of B. subtilis and gives rise to an accumulation of UDP-N-acetyl-muramyl pentapeptide. However, in vitro peptidoglycan synthesis using a particulate membrane preparation is not inhibited by clofoctol. These results suggest that clofoctol does not inhibit a specific enzyme of the cell wall synthesis, and that the inhibition of the cell wall synthesis might be a secondary effect due to a disturbance of the cytoplasmic membrane.

[Clofoctol binding by the bacteria (author's transl)]. / Fixation du clofoctol par les bactéries.

Clofoctol is an antibacterial agent which is active only on Gram + bacteria. The clofoctol binding on bacteria is fast and the number of molecules bound is about 10(6) to 1,5 10(7) molecules per bacteria. Bacteria sensitivity towards clofoctol is the result of their ability to bind with it. Gram negative and Gram + protoplasts of bacteria bind the clofoctol. Binding of clofoctol by B. subtilis is obtained with viable cells and also with thermal inactivated cells. Binding of clofoctol by the cells is reversible and we showed that the 14C clofoctol used for the experiments and the unlabelled clofoctol have the same behaviour. ions as K+, Mg++, Ca++, Mn++, and Fe++, pH variation between 2 to 9, and urea have no influence on the binding of clofoctol by the bacteria, but, sodium lauryl sulfate is an inhibitor of the binding. These results mean that clofoctol bounds are made by hydrophobic links.